In mammalian cells, the structural integrity of the nucleus is conferred by A- and B-type lamins, a meshwork of proteins that underlies the nuclear envelope and forms the nuclear lamina. Mutations scattered along Lmna, which encodes A-type lamins, are associated to a broad range of human diseases, collectively called laminopathies. The molecular etiology of these diseases remains unknown. In mammalian cells, the recent characterization of the LINC complex, an evolutionary-conserved protein complex that spans the nuclear envelope and physically connects the nuclear lamina to the cytoskeleton of mammalian cells suggest that nucleus is intimately tethered to the cytoskeleton. Based on our preliminary results and from data available from lower organisms, we hypothesize that the deleterious effect of Lmna mutations consist in the severe disruption of physical connections that mediate essential physiological cellular processes such as nuclear dynamics, cellular mechanical stiffness and polarization. Three specific aims will be developed in this project: 1) Using live-cell microscopy and a flow-based assay, the rates of translocation of both the nucleus and the microtubule organizing center (MTOC) as well as the MTOC/nucleus distance in real time and in 3D will be compared in mouse embryonic fibroblasts lacking A-type lamins (derived from a mouse model of human muscular dystrophy and cardiomyopathy) and their wild-type counterparts. Using quantitative imaging, we also will determine whether A-type lamin deficiency affects key cytoskeleton-based cell functions, including single-cell motility and MTOC polarization. 2) Based on the hypothesis that the LINC complex mediates several physiological processes controlled by A-type lamins, the contribution of the whole LINC complex as well as of its respective components to cellular mechanical stiffness and cytoskeleton-based cell functions will also be investigated. 3) The effect of disease-associated mutations of Lmna on the LINC complex integrity and cytoskeleton functions will be examined in mouse fibroblasts and myoblasts. We anticipate that this project will shed light on the biophysical principles that govern nucleus dynamics and nucleus-cytoskeleton connections, and will identify key molecular linkers that regulate this interconnection and play a critical role in cellular mechanical stiffness, polarization and motility. Results from experiments using our quantitative assays may also help establish a biophysical basis for the wide variety of disease phenotypes associated to human laminopathies. Public Health Relevance: Mutations scattered along the Lmna gene, which encodes A-type lamins, are associated to a broad range of human diseases, collectively called laminopathies. The proposed research drawing from bioengineering and cell biology may help establish a biophysical basis for the wide variety of disease phenotypes associated to human laminopathies.